The invention relates to a process for the microbiological preparation of aldose-1-epimerase (mutarotase) by cultivating a microorganism, suitable for the formation of this enzyme, of the genus Acinetobacter.
Mutarotase (EC 5.1.3.3) accelerates the establishment of equilibrium between the .alpha.- and .beta.-anomers of aldohexoses, for example between .alpha.- and .beta.-glucose or .alpha.- and .beta.-galactose. The main application of the enzyme is in analytical biochemistry for the acceleration of enzymatic detection reactions for aldoses by means of enzymes specific for the .alpha.- or .beta.-form, where the establishment of equilibrium between the two anomers is the rate-determining step, for example in determination methods with glucose-dehydrogenase, glucose-oxidase, galactose-dehydrogenase or galactose-oxidase. Such determinations are conventional and are described in U.S. Pat. No. 3,964,974 and Anal. Biochem. 43. 312 (1971), which disclosure is incorporated by reference herein. A large-scale industrial application could become a interest, for example, for the glucoamylase/glucose-isomerase process, because glucoamylase releases .beta.-glucose which can be converted by the glucose-isomerase only after mutarotation to the .alpha.-form.
Mutarotase is very widespread in nature. It occurs in various microorganisms (bacteria, yeasts, thread fungi), in plants and in animal tissues.
Significant enzyme contents which allow isolation of mutarotase on an industrial scale have so far been found only in the kidneys of mammals (cattle, pigs); all the known commercial products are prepared from kidneys. It is known from Bailey, Meth. Enzymol. 1975, page 478, that bovine kidneys contain more than 60 times the activity per g of fresh weight than, for example, Escherichia coli. In Biochim. Biophys. Acta 662, 285 (1981), a process for the microbiological preparation of mutarotase from strains of Aspergillus niger is described for the first time. According to this, a mutarotase activity of 4.4 mU/ml of culture broth was obtained from the best strain, the Michaelis constant was 50 mM, and the pH optimum was in the range 5-7.
However, the known microbial process has a very low yield as compared with the preparation process for mutarotase from bovine kidneys. In addition, the characteristics of the enzyme from Aspergillus niger are very unfavorable for the establishment of equilibrium in enzymatic determinations of aldoses.